Reverse genetics permits the recombinant expression and manipulation of viruses in cell culture. It is a powerful tool in virology and vaccine manufacture because it allows rapid production and/or mutation of viruses, including reassortant production. The method involves transfecting host cells with one or more plasmids which encode the viral genome then isolating (or “rescuing”) virus from the cells. It can be used for the production of a wide variety of RNA viruses, including positive-strand RNA viruses [1,2], negative-strand RNA viruses [3,4] and double-stranded RNA viruses [5].
A drawback of known methods is that they rely on plasmids: Generating these plasmids requires cloning steps to be performed in bacteria, which can take several days or weeks to perform and verify for a segmented RNA virus. Such delays interfere with the timetable for yearly production of seasonal influenza vaccines and also prevent a rapid response to a pandemic outbreak. Furthermore, the use of bacteria entails the risk that bacterial contaminants might be introduced when the plasmids are used to transfect a host cell for virus production. These drawbacks are addressed in reference 6 by using linear expression constructs instead of plasmids. The linear expression constructs do not contain amplification and/or selection sequences which are used during bacterial propagation and almost always results in the molecular cloning of a single representative of a viral quasispecies. Such linear expression constructs can be used to transfect host cells directly, giving a much more rapid reverse genetics system: reference 6 suggests that transfection of the linear constructs can be achieved within hours of receiving a viral isolate, avoiding the time required for molecular cloning and allowing access to useful members of the original viral quasispecies population.